5.2.1 Polymerase-Chain-Reaction (PCR)
DNA fragments are generated by PCR using appropriate oligonucleotides (primers).
For cloning strategies, the following protocol was used:
Reaction mix Enzyme mix
Reagent Volume Reagent Volume
H2O 32 μl H2O 8 μl
10x buffer(+MgCl2) 4 μl 10x buffer(+MgCl2) 1 μl
dNTPs (20 mM) 1 μl Pfu polymerase 0.5 μl
Forward Primer (10 pmol) 1 μl Taq polymerase 0.5 μl Reverse Primer (10 pmol) 1 μl
Template 1 μl(100ng)
For amplification in the thermocycler the following program was used:
1) Hot start 94°C 3 min pause (addition of the enzyme mix) 2) Denaturation 94°C 20seconds
3) Hybridization 55-64°C 20seconds
4) Elongation 72°C depending on fragment size (Taq 1000bp/min; Pfu 750bp/min) 5) 30 cycles of steps 2-4
6) Elongation 72°C 5-10min 7) Store at 4°C forever
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Afterwards, PCR product is purified for the further cloning strategy.
For examination of recombinant products, the following protocol is used:
Reaction mix Enzyme mix
Reagent Volume Reagent Volume
H2O 12.3 μl H2O 4.3 μl
10x buffer(+MgCl2) 1.5 μl 10x buffer(+MgCl2) 0.5μl Forward Primer (10 pmol) 0.4 μl Taq polymerase 0.2 μl Reverse Primer (10 pmol) 0.4 μl
Template 0.2μl(10ng)
For amplification in the thermocycler, the following program is used:
1) Hot start 94°C 3 min pause (addition of the enzyme mix) 2) Denaturation 94°C 20seconds
3) Hybridization 55°C 20seconds
4) Elongation 72°C depending on fragment size (Taq 1000bp/min; Pfu 750bp/min) 5) 20 repetitions of steps 2-4
6) Elongation 72°C 5-10min 7) Store at 4°C forever
The PCR products are determined by agarose gel electrophoresis.
5.2.2 Agarose Gel Electrophoresis
DNA fragments can be separated and identified by agarose gel electrophoresis. The separation of the fragments in the gel matrix is dependent on their size, whereas the velocity is proportional to the logarithm of their size. For an optimal separation, the
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concentration of agarose in the gel is chosen dependently on the size of the fragments.
The suitable amount of agarose is solved in 1X TAE buffer by heating and poured into the gel chamber after cooling down to about 50°C. Slots are formed by a comb. The DNA samples are mixed with GEBS, applied to the slots and then separated in 1X TAE at 90 V. A DNA standard (DNA Ladder, Fermentas) is also applied in one slot to facilitate a direct comparison of the fragment sizes. DNA bands are visualized by staining the gel in the Gelred bath, as after intercalation into the DNA, Gelred fluoresces under UV light (λ = 300 nm).
5.2.3 Purification of PCR products
After PCR, the obtained products are purified by QIAquick Gel Extraction Kit (QIAgen), following the provided manufacturers protocol. The concentration is measured by NanoDrop spectrophotometer and the correct size and quantity is determined by agarose-gel-electrophoresis.
5.2.4 Gel-Extraction
DNA fragments analyzed by agarose gel electrophoresis were cut out under UV light.
Afterwards the fragment is purified by QIAquick Gel Extraction Kit (QIAgen), following the provided manufacturers protocol. The concentration of the DNA in elution buffer is determined with NanoDrop spectrophotometer.
5.2.5 Restrictional Digestion
DNA fragments generated by PCR or vectors are digested by restriction enzymes, then can be used for further ligation or analysis. For digestion reaction, mix is prepared using following protocol:
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2 μg plasmid DNA (depending on concentration) 1 μl 10x restriction buffer
0.5 μl restriction enzyme (2 U) ad 10 μl ddH2O
The reaction mix is incubated for 3 h at 37°C. Afterwards the digestion mix was analyzed via agarose gel electrophoreses. The optimal temperatures and buffers for the digestion could be extracted from the enzyme lists provided by the manufacturers (NEB and Fermentas). For the double digestions, the same protocol is used, however, an optimal buffer has to be found.
5.2.6 Ligation
The method of ligation is used to integrate an insert of interest into a plasmid vector.
The following reaction mix is prepared:
x μl plasmid vector x μl insert
1 μl T4-ligase
1 μl 10x T4-ligase buffer ad 10 μl ddH2O
Normally a vector to insert molar ratio of 1:1 to 1:10 is used. The molarity is calculated using following formula:
pmol/μl =DNA(pg/μl)/(660*size[bp])
The reaction mix is incubated at 4°C overnight. Subsequently, the sample is directly transformed into competent E. coli NovaBlue, which means the transformation is performed.
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5.2.7 Cre/lox Recombination
For Cre/lox recombination, the following reaction mix is prepared:
1 μl 10x Cre-recombination buffer 6 μl ddH2O
1 μl (100ng) donor vector 1 μl (100ng) acceptor vector 1 μl Cre-recombinase
The reaction mix is incubated for 45 min at 37°C. Heat inactivation of the enzyme is followed by 70°C for 10 min. Subsequently, the reaction mix is used for transformation in competent E. coli and plated on LB-plates with chloramphenicol and 7% sucrose.
With chloramphenicol and sucrose, it is possible to select desired recombinant colonies that contain a directionally correct gene insert. Clones containing the remaining donor vector, without the gene insert, will express SacB, and therefore, cannot be grown on media containing sucrose. Putative positive clones are verified by restriction digestion and sequencing.
5.2.8 shRNA Construction and Cloning
For the generation of recombinant and shRNA-expressing lentiviral particles, the shRNA vector system pLKO.1 developed by Stewart and colleagues (Stewart et al.
2003) was applied. The different shRNA were designed by using the AAN19 algorithm and a siRNA selection program of the Whitehead Institute for Biomedical Research (http://jura.wi.mit.edu/bioc/siRNAext/). According to the prediction of the siRNA selection program two complementary primers were synthesized. The primers were annealed and cloned via AgeI and EcoRI restriction sites into pLKO.1 (pLKO.1 was a
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gift from Bob Weinberg). The correct insertion of the shRNA cassette was verified by sequencing.
5.2.9 Cloning of gRNA-oligo into pBluescript-U6-MCS and PX458
After the design and synthesis of gRNA-oligos for CRISPR-cas9, prepare the annealing mix in PCR reaction tubes for annealing reaction: 1μl sense primer (100μM), 1μl antisense primer (100μM), 5μl NEB buffer2 and 43μl ddH2O. Annealing reaction is carried out in thermocycler with the following program: 95°C for 4min, start loop, 54x 94°C, 1 min(-1°C/min), close loop, store at 4°C.
Meanwhile, BbsI digested vector is under preparation. 3μg vector is digested with 2μl BbsI. After digestion, treated vector is purified via agarose gel electrophoresis and gel extraction. After the annealing reaction and preparation of digested vector, ligation reaction is performed: 1μl digested vector (about 100ng), 1μl annealing product, 1μl T4 ligase, 1μl T4 ligase buffer and 6μl ddH2O, incubate at 4°C overnight. Next day, T4 ligase is inactivated at 75°C for 10min. Then, BbsI digestion is performed again to destroy the re-ligated vector, by addition of 1μl BbsI and incubation at 37°C for 30min.
Afterward, transformation into E.coli Novablue is performed. With isolated plasmid, PCR reaction and sequencing are carried out to verify the positive colonies.
5.2.10 Transformation of Bacteria
Competent bacteria are thawed on ice, then mixed with the cre-lox recombination sample and incubated for 30min on ice. The heat shock at 42°C for 75sec followed.
Finally the sample is cooled on ice for 5 min and mixed with 1ml LB-medium. The sample is then incubated for 1 h at 37°C on a shaker. After centrifugation at 5000 rpm
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for 3 min, supernatant is discarded and the pellet is re-suspended in the residual medium (approximately 100 μl), which is plated on adequate selective medium plates and incubated over night at 37°C. Next day, from grown colonies, clones are selected, streaked on new selective medium plates and incubated under the same conditions.
5.2.11 DNA Preparation
For the analysis of plasmids after recombination, the plasmids are isolated using the protocol of Birnboim and Doly (Birnboim and Doly, 1979). For high quality plasmids that are used for further processing, e.g. transfection or sequencing, plasmids are prepared with a commercial kit (QIAprep Spin Miniprep Kit, Qiagen), following the provided manufacturer’s protocol. For high quantity of plasmids, a commercial kit (QIAprep Spin Midiprep Kit, Quiagen) is used, following the provided manufacturer´s protocol. The plasmid concentration is determined photometrically via NanoDrop spectrophotometer. The plasmid quality and size are determined by agarose gel electrophoresis.
5.2.12 qRT-PCR evaluation of mRNA levels
Isolation of total RNA is performed according to the procedure with RNeasy Mini Kit.
The concentration of total RNA is measured by Nanodrop and 2 μg of total RNA is applied for the agarose gel (1%) electrophoresis. If two bands are clearly observed without strong degradation, 1-2 μg of total RNA is applied for the reverse transcription using the following procedure: 4 µl 5x RT-buffer, 2 µl dNTP-mix, 1 µl reverse transcriptase, 1.25 µl random hexamer primer (160 µM), 0.1 µl oligo dT18 (100 µM), 1-2 µg RNA, 0.5 µl RNase inhibitor, add up to 20 µl with RNase-free dd H2O, then
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incubate for 1h at 37°C and heat-inactivate at 70°C for 15 min.
Quantitative real-time PCR is conducted with the sensiMixPlus SYBR Kit with the following cycle conditions: 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. Relative expression of target genes is normalized by housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fold change of mRNA levels of target genes is calculated using the formula described by Thomas D Schmittgen and Kenneth J Livak (Schmittgen, Thomas D., and Kenneth J.
Livak. Nature protocols, 2008).
5.2.13 SDS-PAGE (SDS Poly Acrylamide Gel Electrophoresis)
ForSDS-PAGE, gel-casting system and the chamber from BioRad are used. Separation gels have the poly-acrylamid concentration ranging from 8 to 15%, according to the protein size. After addition of TEMED and APS, the separation gel is cast between two glass plates and covered with a layer of ddH2O or isopropanol. After polymerization the water or isopropanol is removed and the stacking gel is cast above the separation gel. Before polymerization a comb is inserted between the glass plates. The completely polymerized gel is put into a gel chamber after removal of the comb and covered with running buffer. Protein samples are mixed with 2x SDS or 4xSDS buffer and boiled for 5 min at 95°C. Subsequently they are loaded into the SDS gel lanes. Protein separation is carried out at 120 V for 1-2 h.
The preparation of separation and stacking gel is performed as follow:
Separation Gel (For one gel)
7% 8% 10% 12.5% 15% 18%
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Acrylamide(40%) 1.75ml 2ml 2.5ml 3.1ml 3.75ml 4.5ml
Water 5.75ml 5.5ml 5.0ml 4.3ml 3.65ml 2.9ml
1.5M Tris PH8.8 2.5ml 2.5ml 2.5ml 2.5ml 2.5ml 2.5ml Degas!!!
20% SDS 50μl 50μl 50μl 50μl 50μl 50μl
10% APS 30μl 30μl 30μl 30μl 30μl 30μl
TEMED 15μl 15μl 15μl 15μl 15μl 15μl
Stacking Gel (For two gels) 5%
Acrylamide(40%) 1.25ml
Water 6.15ml
0.5M Tris PH6.8 2.5ml Degas!!!
20% SDS 50μl
10% APS 30μl
TEMED 15μl
5.2.14 Western blotting (wet-blot)
Once the SDS PAGE is done, prepare 6 pieces of thick filter paper and one PVDF membrane to the dimensions of the gel. The membrane is wetted in methanol for 30 seconds and equilibrated in transfer buffer. The right/top corner of the membrane is cut for labelling. To assemble the transfer sandwich, the sponge is placed on the bottom (anode), then 3 pieces of thick filter paper, the membrane, the gel, the 3 pieces of thick
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filter paper, and finally, the sponge on top (cathode). Once the stacks are positioned in the chamber, place the lid and start running at 30V overnight or 150 mA for 2h.